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Journal of Virology, November 1999, p. 9673-9678, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Trophoblastic Epithelial Barrier Is Not Infected in Full-Term
Placentae of Human Immunodeficiency Virus-Seropositive Mothers
Undergoing Antiretroviral Therapy
Charlotte
Tscherning-Casper,1
Nikos
Papadogiannakis,2
Maria
Anvret,3,4
Lisa
Stolpe,3
Susanne
Lindgren,5
Ann Britt
Bohlin,6
Jan
Albert,7 and
Eva Maria
Fenyö1,*
Microbiology and Tumorbiology Center,
Karolinska Institute,1 Departments of
Pathology,2 Obstetrics and
Gynecology,5 and
Pediatrics,6 Huddinge Hospital,
Karolinska Institute, Department of Clinical
Genetics,3 and Department of
Clinical Neuroscience,4 Karolinska Hospital, and
Swedish Institute for Infectious Disease
Control,7 Stockholm, Sweden
Received 14 August 1998/Accepted 9 July 1999
 |
ABSTRACT |
To study the mechanism of the placental barrier function, we
examined 10 matched samples of term placentae, cord blood, and maternal
blood obtained at delivery from human immunodeficiency virus
(HIV)-infected mothers with children diagnosed as HIV negative in
Sweden. All placentae were histologically normal, and immunochemistry for HIV type 1 p24 and gp120 antigens was negative. Highly purified trophoblasts (93 to 99% purity) were negative for HIV DNA and RNA,
indicating that the trophoblasts were uninfected. Although HIV DNA was
detected in placenta-derived T lymphocytes and monocytes, microsatellite analysis showed that these cells were a mixture of
maternal and fetal cells. Our study indicates that the placental barrier, i.e., the trophoblastic layer, is not HIV infected and, consequently, HIV infection of the fetus is likely to occur through other routes, such as breaks in the placental barrier.
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TEXT |
Maternal-infant transmission of
human immunodeficiency virus type 1 (HIV-1) is the primary cause of
HIV-1 infection in children. The risk of mother-to-child transmission
of HIV ranges from about 15% in Europe (12, 13) to 39% in
Africa (35). The AIDS Clinical Trial Group Protocol 076 (ACTG076) demonstrated that a regimen of zidovudine to a selected group
of HIV-infected pregnant women during the second and third trimesters
of pregnancy along with administration of zidovudine to their newborns
reduced the risk of perinatal transmission by two-thirds
(7). This study demonstrated for the first time that
perinatal HIV transmission can be prevented. However, this treatment is
expensive and is not available in many parts of the world, such as
sub-Saharan Africa and developing areas of the Americas where more than
three-quarters of the perinatally infected children live
(36). Available data suggest that most perinatal HIV
transmissions occur at or just before delivery (5, 11, 14,
19), but cases of intrauterine transmission (8, 24, 37,
40) and transmission by breastfeeding (41) have also
been described. It is important to obtain more detailed information about the timing and the mechanisms of perinatal HIV transmission, because such knowledge is likely to have a significant effect on future
intervention strategies aiming at prevention of maternofetal transmission.
The placenta provides a potential barrier between the maternal and
fetal circulations, but limited attention has been given to its role in
the transmission of HIV. The human placenta is of the villous
hemochorial type and consists of a vast array of fetal villi, which are
bathed directly in the circulating maternal blood (17). At
the end of the pregnancy, the villi represent a surface area of 10 to
14 m2 and therefore permit extensive and intimate contact
between fetal tissues and maternal blood. The outermost layer of the
villi consists of syncytiotrophoblasts, which form a continuous
multinucleated epithelium generated from and maintained by an
underlying population of mononuclear cytotrophoblast cells. This
trophoblastic layer is supported by a basement membrane, which
separates it from the mesenchymal cells of the villous core. Within the
core are the fetal capillaries and a significant number of macrophages,
often referred to as Hofbauer cells, some of which express the CD4
molecule (30). Since trophoblastic cells constitute the
external layer of chorionic villi, they are in direct contact with
maternal blood which makes them a primary target for maternal
blood-borne infections. However, the placental barrier is not complete,
and there is evidence that bidirectional traffic of cells, including
leukocytes, may occur in human pregnancy (32).
To determine if cells in the full-term placentae of HIV-seropositive
mothers are infected, and if so, which cell type is affected, we
prospectively collected 10 term placentae from HIV-infected mothers (9 infected with HIV-1 and 1 infected with HIV-2) from delivery wards in
the Stockholm area (Sweden) between October 1996 and November 1997. These women were participants in a larger prospective multicenter study
which evaluated factors influencing maternofetal transmission of HIV
(13). Ethical approval for the study was obtained, along
with informed consent from all women. Four mothers were infected with
HIV-1 of genetic subtype C, three with subtype A, and two with subtype
B (Table 1). Delivery occurred at term in
all cases. Elective cesarean section was performed in six cases and
vaginal delivery in four cases. One of these ended in emergency
cesarean section because of signs of fetal asphyxia. Nine infants, more
than 18 months old, have seroreverted and are uninfected. One child
died of sudden infant death syndrome at 1 month of age. Virus isolation
and DNA PCR using peripheral blood mononuclear cells (PBMC) as well as
mesenteric lymph nodes of this child were negative for HIV-1.
Isolation of placental trophoblastic cells.
Macroscopic and
routine histologic examination of all freshly delivered placentae,
extraplacental fetal membranes, and the umbilical cord did not reveal
any gross abnormalities in any of the cases. Immunohistochemical
examination of formalin-fixed, paraffin wax-embedded tissue did not
detect HIV-1 p24 and gp120 antigen. Enriched trophoblastic cell
populations were then isolated by using the method of Kliman et al.
(20). Briefly, placental tissue was extensively washed with
Hanks balanced salt solution (HBSS) (GibcoBRL, Life Technologies),
minced, and digested with trypsin (Difco, Detroit, Mich.) and DNase I
type IV (Sigma). The resulting cell suspensions were collected and
subjected to discontinuous Percoll (Pharmacia) density gradient
centrifugation at 1,200 × g at room temperature for 20 min to enrich for trophoblasts. The mononuclear cell fraction was
recovered and washed twice with cold HBSS. This enriched trophoblastic
cell preparation contained residual contaminating cells, mainly
macrophages expressing CD14, granulocytes expressing CD45, endothelial
cells expressing CD31, and blood elements such as T lymphocytes
expressing CD3 (9).
The purity of trophoblasts was further increased by adding a new
immunomagnetic purification step. Thus, highly purified trophoblasts were obtained by removal of contaminating cells from the enriched trophoblast preparations with immunomagnetic beads (Dynabeads M-450;
Dynal, Oslo, Norway) coated with different monoclonal antibodies. By
successive immunoelimination, we removed Hofbauer cells, monocytes and
macrophages (mouse anti-human CD14, TÜK4; Dakopatts AB), and T
lymphocytes (mouse anti-human CD3, pure Leu4; Becton Dickinson). Thereafter, in a third step, granulocytic cells (mouse anti-human CD45,
T29/33; Dakopatts AB), endothelial cells (mouse anti-human CD31,
JC/70A; Dakopatts AB), and the remaining macrophages and T lymphocytes
were simultaneously removed.
The purity of the enriched and purified trophoblasts was evaluated by
microscopic examination for morphology, as well as by
flow cytometry
performed after staining with mouse monoclonal
antibodies GB25 raised
against placental cyto- and syncytiotrophoblasts
of the chorionic villi
from full-term placentae (
15) and GB17
raised against
syncytiotrophoblasts of the chorionic villi from
term and from first
and second trimester placentae (
16) and
with a rabbit
F(ab')
2 anti-mouse immunoglobulin G fluorescein
isothiocyanate-conjugated second antibody (Dakopatts AB). GB25
and GB17
were produced by hybridoma cells obtained following immunization
of
mice with isolated human term microvilli (
15,
16) and were
a
gift from Gerard Chaouat (Hôpital Antoine-Béclère,
Clamart,
France). In addition, the presence of contaminating cells was
evaluated with flow cytometry using monoclonal antibodies directed
against CD3, CD14, CD45, CD31, and CD4 (CD4 BL4 pure; Immunotech,
Marseille,
France).
The trophoblastic cell preparations consisted mainly of
cytotrophoblasts with high expression of GB25 and low expression of
GB17 (data not shown). The mean (± standard error of the mean)
percentage of trophoblastic cells expressing GB25 in the enriched
(undepleted) cell preparations was 88.7 ± 6.6% and that in the
purified depleted cell preparations was 95.4 ± 2.2%. Table
2 shows the purity of the enriched and
purified trophoblastic cell
preparations from the 10 placentae. The
levels of contaminating
cells, mainly T lymphocytes and macrophages,
were monitored in
the same way and were between 1 and 10% (Table
2).
Virus isolation and detection of HIV DNA and RNA.
Virus
isolation (23, 39) was attempted from extensively washed
chorionic villous tissues of placentae 1, 2, 4, 5, and 6, from fetal
membranes of placentae 4, 5, and 6, and from the CD14+
fractions of the enriched (undepleted) trophoblastic cell populations of placentae 2, 4, and 5. With the exception of the
CD3+/CD14+ fraction of placenta 1, none of the
cultures was virus isolation positive. In view of the very low viral
loads and negative virus isolations even from the mothers' PBMC, it is
not surprising that no virus could be recovered from placental cells.
This suggested that there is very little, if any virus present in the
placentae studied. Therefore, we performed PCR for detection of HIV DNA in the different placental cell preparations as well as the mothers' PBMC and the cord blood samples (CBMC). For PCR, cells were lysed (4) and nested PCR was performed in duplicate on 10 µl of
cell lysate corresponding to 105 cells, using primers
JA79-JA82 and JA80-JA81 (1, 25), known to amplify diverse
genetic subtypes within group M of HIV-1 (25). For
HIV-2-infected patient 3, the primer sets JA47-JA52 and JA48-JA49 (pol) were used (33). The amplimers were
visualized by ethidium bromide staining after electrophoresis in a
1.5% agarose gel. For each amplification, a negative control without
DNA and a positive control consisting of 10 DNA copies of
HIV-1MN or HIV-2K135 (3) were
diluted into an HIV-negative PBMC lysate corresponding to 105 cells. A minimum of two, but usually more, independent
PCRs were performed on cell preparations with an initially negative PCR result. In addition, each sample was tested for the presence of human
DNA by using the primer set PCO3-PCO4 detecting 
-globin (31).
PCR-positive cell fractions were further analyzed by semiquantitative
limiting dilution PCR analysis as described previously
(
4).
Briefly, a fivefold dilution series was prepared from
each PCR-positive
cell fraction, and at least five independent
PCRs were performed on
aliquots containing 1 × 10
5, 2 × 10
4, 4 × 10
3, and 8 × 10
2 cell equivalents of DNA. The number of HIV DNA copies
per 100,000
cells was determined by using the Poisson distribution
formula,
according to which the precursor frequency (or DNA copy
number)
is the inverse fraction of the number of cell equivalents
required
to give 63% positive reactions (
4).
The mothers' PBMC were PCR positive, indicating that the primers used
had the ability to amplify the DNA of the virus variants
carried by
these mothers (Table
3). The
corresponding cord blood
samples were all PCR negative. The enriched
(undepleted) trophoblastic
cell preparations from all 10 placentae were
PCR positive, whereas
all purified trophoblastic cell preparations were
regularly negative
(Table
3). In addition, the CD3
+ cell
fractions from all except two mothers were PCR positive,
whereas most
CD14
+ cell fractions were negative. These results indicated
that the
HIV DNA sequences detected in the enriched (undepleted)
trophoblastic
cell preparations originated from contaminating
CD3
+ and CD14
+ cells, since removal of these
cells resulted in the loss of a
PCR signal in the purified trophoblast
preparation. For HIV-2-positive
placenta 3 and for placenta 4, the weak
PCR signal was lost during
the cell fractionations (Table
3).
The enriched, but undepleted, trophoblastic cell preparations had lower
HIV DNA levels than the corresponding PBMC. Similarly,
the enriched
(undepleted) trophoblasts had lower HIV DNA copy
numbers than the
CD3
+ placental cell fraction in six out of the seven cases
for which
comparison was possible (Table
3). In fact, in most cases the
HIV DNA copy numbers in CD3
+ placental cells were
comparable to those of PBMC. This indicates
that the enriched
(undepleted) trophoblast preparations carry
HIV DNA due to the
remaining CD3
+ cells. The HIV DNA levels in PBMC or
CD3
+ placental cells showed no clear correlation with
CD4
+ lymphocyte counts or plasma HIV RNA levels of the
mothers.
The purified (depleted) trophoblastic cells isolated from
HIV-1-positive placentae were also screened for the presence of
HIV-1
RNA by using reverse transcription-PCR (RT-PCR). Briefly,
total RNA was
extracted from purified trophoblastic cells using
the Nuclisens method
according to the manufacturer's recommendation
(Organon Teknika,
Boxtel, The Netherlands) and reverse transcribed
into cDNA
(first-strand cDNA synthesis kit; Amersham Pharmacia
Biotec, Uppsala,
Sweden). Detection of HIV RNA was performed by
using the previously
described
pol primers JA79-JA82 (
1,
25)
and was
negative for all placentae. The efficiency of this method
was tested on
blood donor PBMC spiked with a known amount of viral
RNA. These
experiments showed that 30% of the RNA was successfully
extracted,
reverse transcribed, and PCR amplified (data not
shown).
Genomic microsatellite analysis.
Genomic microsatellite
analysis was used to determine the origin of the different cell types
isolated from the placentae. By this method, cells from different
individuals, such as a mother and her semiallogeneic child, can be
distinguished by analysis of hypervariable regions of the human genome.
The genotypes of the mother and the corresponding infant were
determined by analysis of multiple loci by PCR amplification using
33P-labelled dATPs for the loci D1S547, D1S1677, D1S1679,
D1S2134, and RB20.1 or the 6-carboxyl-fluorescein
(FAM)-labelled primers D5S346 (LNS-CA repeat marker) and D4S127
(CAG repeat in the Huntington disease gene) (38). The
maternal and fetal cells were informative (heterozygous) for the
respective microsatellite markers, so that the alleles could be
identified (illustrated in Fig. 1). The
different placental preparations were then also subjected to genomic
microsatellite analyses of alleles which differed between the
respective mother and infant (listed for each sample in Table
4). This allowed us to evaluate if the
different cell fractions in the placenta were of maternal or fetal
origin. We found that CD3+ and CD14+ cells from
all the placentae were mixtures of maternal and fetal cells as
demonstrated by the presence of three different alleles (Table 4). For
placentae 3 and 4, there was not enough material from the
CD3+ and CD14+ cell fraction to perform this
analysis. Nevertheless, the enriched mixed trophoblastic cell
populations from these placentae were shown to be of fetal origin. This
finding indicates that microsatellite analysis does not detect small
amounts of maternal contamination, i.e., contaminating CD3+
and CD14+ cells (6).
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FIG. 1.
Origin of different cell populations in maternal blood
(a), cord blood (b), placental T lymphocytes (c), and placental
macrophages (d) in mother 6 using microsatellite analysis with
noninformative marker D5S346 (A) and informative marker D4S127 (B). See
Table 4, footnote b, for an explanation of the numbers 1, 2, and 3.
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View this table:
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TABLE 4.
Origin of different cell populations in the placenta,
cord blood, and maternal blood by genomic microsatellite analysis
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In the present study, we show that highly purified primary placental
trophoblasts obtained from HIV-seropositive mothers are
negative upon
PCR for HIV DNA and RNA. Our results demonstrate,
for the first time,
that villous trophoblasts in term placentae
of mothers undergoing
antiviral therapy are uninfected. These
results are at variance with
those of Lee et al. (
22), who detected
HIV
gag
sequences by RT-PCR in placental cell preparations from
30 HIV-seropositive mothers, of whom 23 were undergoing antiretroviral
therapy throughout pregnancy and/or intrapartum. Lee et al.
(
22)
obtained placental cells after only one round of
immunomagnetic
cell depletion using a single anti-CD45 monoclonal
antibody. The
purity of these trophoblast preparations was determined
solely
by morphology and was estimated to be on average 95.8%. As
estimated
by microscopy, contaminating leukocytes, macrophages, and
granulocytes
were present in less than 3%. In contrast, we
carried out immunomagnetic
cell separations in three sequential steps
and used specific monoclonal
antibodies for each cell type, such as
placental macrophages or
Hofbauer cells and blood monocytes (CD14), T
lymphocytes (CD3),
and residual leukocytes (CD45). In addition, the
placental cell
preparations were monitored at each step of
immunomagnetic cell
separation for the presence of trophoblast membrane
antigen GB25.
The purity of our enriched (through Percoll gradient
only) trophoblast
preparations was comparable to those of other studies
(
22,
29),
and indeed HIV DNA could be amplified from all
these trophoblast
preparations. However and importantly, HIV DNA was
not detected
in our immunomagnetically purified trophoblast
preparations. In
fact, we were able to identify the HIV-positive cells
as CD3
+ or CD14
+, that is, belonging to the
fraction of contaminating T lymphocytes
and macrophages. This is in
line with the results of previous
studies demonstrating that
nontrophoblastic placental cells carry
HIV infection in vivo and are
susceptible to infection in vitro
(
2,
26-28). Taken
together, the cell fractionation procedures
whereby trophoblasts are
obtained seem to be crucial (
9) and
the level of
contaminating nontrophoblastic cells will strongly
influence the
outcome of HIV detection. Our results show that
the highly purified
trophoblasts are uninfected. This conclusion
is supported by the
findings of Kilani et al. (
18), who found
that pure
placental trophoblasts resist infection by multiple
cell-free primary
HIV-1 isolates. In addition, the purified trophoblasts
did not appear
to express CD4, which is in agreement with previous
findings (
10,
21). These results are further corroborated
by
immunohistochemical data, which failed to detect any HIV-1
p24 or gp120
antigens in the examined placental tissues, including
trophoblasts.
Thus, the trophoblastic epithelium might be a significant
barrier to
transplacental HIV
infection.
Another complicating factor in these types of experiments is the fact
that, due to insufficient separation techniques, the
nontrophoblastic
cell fraction of the placenta consists of a mixture
of maternal and
fetal cells. Consequently, the demonstration of
HIV infection in
nontrophoblastic cells may simply be a reflection
of the fact that the
maternal blood in the placenta contains HIV-infected
CD4
+
lymphocytes and monocytes. In our study, we used two approaches
to test
the origin of HIV-infected cells. First, the level of
HIV DNA in each
cell fraction was evaluated in a semiquantitative,
limiting dilution
assay. For all mothers, PBMC and placental CD3
+ T
lymphocytes carried similar amounts of HIV-DNA, suggesting
that the
viral DNA was of maternal origin. Second, we determined
the origin of
cells in the different cell preparations by microsatellite
analysis
(
6). Indeed, in our experiments we could confirm that
the
different nontrophoblastic placental fractions consisted of
mixtures of
maternal and fetal cells, whereas the trophoblasts
were of fetal
origin.
All mothers in our study were receiving zidovudine therapy according to
the ACTG076 protocol, and some mothers were in addition
receiving other
reverse transcriptase inhibitors. Thus, the study
population was highly
representative of HIV-1-infected mothers
in Europe and in the United
States where most seropositive women
are treated during pregnancy.
Zidovudine undergoes activation
through phosphorylation within
trophoblasts and Hofbauer cells,
but at a rate 50- to 100-fold lower
than in lymphocytes (
34).
Thus, we cannot exclude the
possibility that the absence of HIV-1
infection in the trophoblasts was
influenced by the antiretroviral
therapy. Since in our study all
children appeared to be uninfected,
we cannot formally exclude the
possibility that trophoblasts are
infected in those pregnancies which
result in transmission of
HIV infection to the fetus. However, in the
study of Menu et al.
(
29), in which four women were
transmitting and eight were nontransmitting,
no correlation was found
between the frequency of PCR positivity
of enriched trophoblast cell
preparations and transmission. Nevertheless,
immunomagnetic cell
depletion for further purification of the
trophoblasts was not
performed in this study (
29).
Taken together, our results indicate that the trophoblastic barrier
remains uninfected in full-term placentae of HIV-seropositive
mothers
undergoing antiretroviral therapy. We suggest that in
utero HIV
transmission, if at all, occurs at the end of gestation
through
alternative routes, such as chorioamnionitis with leakage
of the virus
into the amniotic cavity or trophoblast damage. This
knowledge is
important for the design of new, simpler intervention
strategies aiming
at the prevention of mother-to-child transmission
of
HIV.
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ACKNOWLEDGMENTS |
We thank Kajsa Aperia, Ellen Sölver, Elisabet Lilja, AnnaLena
Andersson, Robert Fredriksson, Kerstin Andreasson, and Dalma Vödrös for technical help; Zhiping Zang (CMM, Karolinska
Institute, Stockholm) for technical assistance with the microsatellite
analysis; Gérard Chaouat, Barbara Mognetti (Hopital Antoine
Béclère, Clamart, France) and Elisabeth Menu (Pasteur
Institute, Paris, France) for teaching the placental separation
method and giving the monoclonal antibodies anti-GB25 and anti-GB17; Bo
Anzén (Department of Gynecology and Obstetrics, Danderyd
Hospital, Stockholm) and Bo Möller (Department of Gynecology and
Obstetrics, Mälar Hospital, Eskilstuna) for supply of samples;
Erik Belfrage (Department of Pediatrics, Karolinska Hospital,
Stockholm), Knut Lidman (Department of Infectious Diseases, Danderyd
Hospital, Stockholm), and Ann Charlotte Lindholm (Department of
Infectious Diseases, Mälar Hospital, Eskilstuna) for clinical and
laboratory data; Anneka Ehrnst for discussions; and Olivier Casper for
invaluable technical help and discussion of the research findings.
This work was supported by grants from the European Network for In
Utero Transmission of HIV and the Swedish Medical Research Council.
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FOOTNOTES |
*
Corresponding author. Mailing address: Microbiology and
Tumorbiology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Phone: 4687286323. Fax: 468331399. E-mail:
eva.maria.fenyo{at}mtc.ki.se.
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Journal of Virology, November 1999, p. 9673-9678, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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